Although vaccines represent one of the triumphs of medicine, highly effective vaccines have not yet been developed for many devastating diseases, including malaria, cancer, HIV/AIDs or tuberculosis, nor is there a universal vaccine for influenza. Each of these diseases requires a differently tuned immune response, with unique specificities and phenotypes of T cell memory and antibody responses, a diversity that is difficult to achieve with the limited arsenal of delivery systems and adjuvants that is currently available. This project, which involves a close collaboration between research groups led by a bioengineer and a basic immunologist, will focus on the development of a potentially new vaccine platform that is based on antigenic peptides and proteins that are designed to self-assemble into nanofiber materials. By virtue of their modular non-covalent construction and ability to incorporate a wide variety and dose of different antigens and immunostimulating compounds, we posit that these materials can be quickly and systematically tuned to independently optimize both the strength and phenotype of B cell and T cell responses. Independent tuning of T and B cell responses is more limited for current subunit vaccines with a limited choice of FDA-approved adjuvants, for whole-organism vaccines, or even for other self-assembling systems. Furthermore, we have recently found that peptide nanofibers elicit strong T cells and B cell response, yet they elicit no discernable inflammation at the site of immunization, which is in contrast with other clinical and investigational adjuvants. These nanofibers require signaling through MyD88 for their activity, although the full mechanism of their immunogenicity through this pathway has not yet been elucidated. Accordingly, this project consists of two integrated goals. The first is to elucidate the mechanism of how self- assembled peptide nanofibers stimulate strong immune responses, for T cells (Aim 1) and B cells (Aim 2). The working model of T cell activation is that self-assembling nanofibers stimulate antigen-presenting cells (APCs) via signaling pathways that require the adaptor protein, MyD88, and that they activate only the dendritic cells (DCs) in the draining lymph nodes that acquire the nanofibers. In contrast, particulate adjuvants such as alum activate broader populations of APCs at the injection site. The working model for the stimulation of B cell/antibody responses is that Q11 nanofibers activate complement and initially engage, via complement receptor 2 (CR2), marginal zone B cells that shuttle antigen-Q11 to the B cell follicles where they engage antigen-specific B cells. The second goal is to design new capabilities into the nanofiber system, including a novel means for including folded protein antigens, ways to include specific amounts of selected TLR agonists, and strategies to stimulate CD8+ T cell responses. Combining the new materials and mechanistic insight acquired, we will iteratively refine and test multi-antigen self-assemblies as vaccines against influenza in mice.